Carbohydrate Polymers 125 (2015) 6168

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Characterization of enzymatically modied rice and barley starches

with amylosucrase at scale-up production
Bum-Su Kim a , Hyun-Seok Kim b , Sang-Ho Yoo a,
a
Department of Food Science & Technology and Carbohydrate Bioproduct Research Center, Sejong University, Gunja-dong 98, Gwangjin-gu,
Seoul 143-747, Republic of Korea
b
Department of Food Science and Biotechnology, Andong National University, 1375 Gyeongdong-ro, Andong, Gyeongsangbuk-do 760-749,
Republic of Korea

a r t i c l e

i n f o

Article history:
Received 7 January 2014
Received in revised form 16 February 2015
Accepted 18 February 2015
Available online 5 March 2015
Keywords:
Amylosucrase
Resistant starch
Physicochemical property
Rice
Barley

a b s t r a c t
Physicochemical properties of Neisseria polysaccharea amylosucrase (NpAS)-treated rice and barley
starches were investigated at scale-up production. Pre-gelatinized rice and barley starches were treated
with signicantly lower NpAS dose (0.1 U/mL) but 100 times larger reaction volume (3500 mL), compared
to the analytical scale (35 mL) used in the previous study. NpAS-treated starches in this scale-up production were characterized with respect to reaction efciency (RE), resistant starch (RS) content, amylopectin
(AP) branch-chain length distribution, solubility, swelling power, pasting viscosity, and thermal transition properties. The RE enhanced up to 1.8 times by increasing the reaction volume, which improved
the RS content and AP branch-chain lengths of NpAS-treated starches. Compared with the native starch,
NpAS-treated starches exhibited lower solubility and swelling power, lower pasting viscosity, and a large
increase in the melting peak temperature. Consequently, NpAS treatment of pre-gelatinized starches in
this study would be a potential way of replacing commercial RS production.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Resistant starch (RS) is the indigestible residues of starches
and starch-based products, as native, gelatinized, and retrograded
forms, which are subjected to digestion by amylolytic enzymes during their travel through the gastrointestinal tract (Higgins, 2004).
RS was designed to allow persons suffering from Type II diabetes
and obesity to moderate their glycemic and insulinemic responses
and reduce their intake of sugars resulting from starch digestion
(Higgins, 2004; Kim et al., 2013; Sharma, Singh, & Ritika, 2008).
Recent researches have reported that consumption of RS possessed
physiological health benets: enhancing satiety and bowel health
as dietary ber and/or prebiotics, improving glycemic and insulinemic responses, enhancing body weight loss and maintenance, and
improving blood lipid proles (Higgins, 2004; Lee, Yoo, & Lee, 2012;
Sharma et al., 2008). Thus, RS is emerging as a physiologically benecial food ingredient that prevents metabolic disease and promotes
health.
Currently, commercial RS products have been released in the
market as the forms of the retrograded high-amylose starches

Corresponding author. Tel.: +82 2 3408 3221; fax: +82 2 3408 4319.
E-mail address: shyoo@sejong.ac.kr (S.-H. Yoo).
http://dx.doi.org/10.1016/j.carbpol.2015.02.048
0144-8617/ 2015 Elsevier Ltd. All rights reserved.

and chemically modied starches such as particular phosphorylated cross-linked starches. While the retrograded high-amylose
starches lost some of their resistance to amylolytic enzymes after
heat treatment in aqueous media and high-moisture food systems
(Kim et al., 2010, 2013; Sharma et al., 2008), chemically modied
starches still exhibited enhanced slowly digestible (SDS) and RS
even after being subjected to varied food processes (Kim et al.,
2010). Despite the better resistance of chemically modied starches
to enzymes, many food processors would like to avoid utilization
of chemically modied starches in their products, due to environmental and food safety concerns (Kim & BeMiller, 2012; Villwock
& BeMiller, 2005).
As an alternative approach to preparing RS products, a few
studies attempted to treat partially and/or completely gelatinized
starches with amylosucrase (NpAS) from Neisseria polysaccharea
(Kim et al., 2013; Ryu et al., 2010; Shin, Choi, Park, & Moon,
2010). NpAS sequentially transfers glucosyl units from sucrose
onto pre-existing acceptor molecules (e.g., non-reducing ends of
starch molecules) (Ryu et al., 2010). Thus, NpAS treatment of starch
molecules resulted in the extension of -glucan chains at their nonreducing ends (Kim et al., 2013; Ryu et al., 2010; Shin et al., 2010),
thus lengthening the starch branch-chains and increasing the
weight-average molecular weights and gyration radii of the overall
molecules (Shin et al., 2010). A signicant increase in proportions

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B.-S. Kim et al. / Carbohydrate Polymers 125 (2015) 6168

of intermediate (DP 2536) and longer (DP > 36) amylopectin (AP)
branch-chains was commonly observed for NpAS-treated starches
(Kim et al., 2013; Ryu et al., 2010; Shin et al., 2010). This phenomenon that AP branch-chains were lengthened through NpAS
treatment appeared to be the result of converting natural A-type
crystal packing arrangements for cereal starches (rice, corn) to Btype (Ryu et al., 2010; Shin et al., 2010). RS contents of NpAS-treated
starches (corn, rice, barley) freshly cooked in aqueous media ranged
from 13.6 to 51.8% (based on dry starch weight) (Kim et al., 2013;
Ryu et al., 2010). Also, NpAS-treated starches had higher melting
temperatures (Ryu et al., 2010; Shin et al., 2010), as reported for
highly phosphorylated cross-linked starches possessing greater RS
contents (Chung, Woo, & Lim, 2004; Woo & Seib, 2002). In the studies to date, however, preparation of NpAS-treated starches were
conned to the small scale NpAS reaction involving excessive NpAS
loading. Little information was available on physical and thermal
properties of NpAS-treated starches in contrast to their structural
characteristics and RS contents.
The objectives of this study were to: (1) structurally modify
pre-gelatinized rice and barley starches (with different amylose contents) with the reduced amount of NpAS required per
weight of starch as the reaction volume of the substrate (starch
and sugar) mixture increased, (2) characterize the biocatalytically
transformed starch materials in regard to amylopectin ne structure and RS content to determine if NpAS would function in a
reaction system containing an excess substrate, and (3) further
investigate starch solubility, swelling power, gelatinization, and
pasting viscosity properties which could directly inuence the enduse quality of food products.
2. Materials and methods
2.1. Materials
Rice and barley starches isolated from rice (Goamy, Dongjin,
Manmi, and Sinsunchal varieties) and barley (Gwangan and Chal
varieties) grains in the previous study (Kim et al., 2013) were used
for this study. Goamy, Dongjin, Manmi, and Sinsunchal rice starches
possessed amylose (AM) contents of 27.3%, 18.9%, 11.4%, and 3.6%,
respectively (Kim et al., 2013). AM contents of Gwangan and Chal
barley starches were 29.8% and 6.5%, respectively (Kim et al., 2013).
Other chemicals and reagents were of analytical grades.
2.2. Modication of rice and barley starches with NpAS
NpAS was produced and puried according to procedures outlined by Ryu et al. (2010). NpAS treatment of pre-gelatinized rice
and barley starches was conducted according to the method of Kim
et al. (2013) with some modications while the reaction volume
was increased by 100 times. Starch samples (100 g, dry weight
basis or d.b.) as an acceptor were suspended in 50 mM TrisHCl
buffer (pH 7.0; 3500 mL) containing 300 mM sucrose as a donor.
The starch dispersion was heated at 100 C for 1 h and then, cooled
to room temperature (24 C). This substrate dispersion was transferred into a water-jacketed vessel (5000 mL) with one port lid,
followed by addition of NpAS (0.1 U/mL; total 350 U in the reaction mixture). The reaction was conducted at 35 C for 24 h under
constant stirring (250 rpm) using an overhead stirrer (MS-3060,
Misung Scientic Co. Ltd., Yangju, Korea) equipped with a cross
stirring rod. At 24 h, the reaction mixture was heated at 100 C
for 5 min to inactivate NpAS, and cooled to room temperature.
NpAS-treated starch was recovered by centrifugation (11,000 g,
4 C, 20 min), and the supernatant was taken to determine reaction efciency (RE) of NpAS. The starch pellet was washed four
times with double-distilled water (DDW), freeze-dried, ground, and

passed through a 100-mesh sieve. The starch sample was stored in a

desiccator at room temperature for further analysis. Yields of NpAStreated starches were dened as the percent ratio of the weights
(d.b.) of the insoluble glucans recovered after NpAS reaction to
the initial weights (d.b.) of their corresponding native starches.
Molar concentrations of fructose in the supernatant were determined using high-performance anion-exchange chromatography
(HPAEC) to determine the RE (Wang et al., 2012). RE was calculated as the percent ratio of molar concentrations of fructose in
the supernatant recovered after NpAS reaction to initial sucrose
concentration (300 mM).
2.3. Determination of resistant starch
RS contents of native and NpAS-treated starch samples were
determined by the method of McCleary and Monaghan (2002).
Starch sample (100 mg, d.b.) was dissolved in 100 mM sodium
maleate buffer (pH 6.0, 2 mL) at 95 C for 1 h. Enzyme solution
(2 mL) containing pancreatin and amyloglucosidase was added to
the gelatinized starch solution, and incubated at 37 C for 16 h,
after which the reaction mixture was mixed with absolute ethanol
(4 mL). The supernatant and precipitate were separated by centrifugation (11,000 g, 4 C, 20 min) and set aside, respectively. The
supernatant was adjusted to 100 mL, and an amount of glucose
(equivalent to readily digestible starch) in the diluted supernatant
was determined via the GOPOD method. For insoluble resistant
starch, 2 M KOH (2 mL) was added to the precipitate and dissolved
for 20 min, after which sodium acetate buffer (8 ml, 1.2 M, pH 3.8)
and amyloglucosidase (0.1 mL) were added. The reaction mixture
was incubated at 50 C for 90 min, and the amount of glucose
(equivalent to insoluble resistant starch) in the reaction mixture
was determined by GOPOD. Total RS content was calculated by
subtracting the content of readily digestible starch from the initial
sample weight.
2.4. Amylopectin branch-chain distribution
Starch sample (10 mg, d.b.) was suspended in 90% aqueous
dimethyl sulfoxide (1 mL), and heated in a boiling water bath
for 1 h. Starch solution was mixed with absolute ethanol (6 mL),
and centrifuged at 4500 g for 15 min, after which the supernatant was removed. The precipitate was dissolved again with
pre-heated (96.5 C) 10 mM sodium acetate buffer (pH 3.5, 1 mL).
The solution was then cooled to 40 C, mixed with isoamylase
(0.6 units/mg starch, Hayashibara Biochemical Laboratories, Inc.,
Okayama, Japan), and incubated at 40 C for 48 h. The mixture
was then heated in a boiling water bath for 10 min to inactivate
isoamylase. The debranched starch solution was passed through a
0.45-m syringe lter, and injected onto HPAEC system to examine
AP branch-chain distribution (Ryu et al., 2010). The HPAEC system
(DX-600 series, Dionex, USA) with a pulse amperometric detector
(PAD, Dionex, USA) was equipped with a CarboPac PA-1 column
(4 250 mm, Dionex, USA). Separation was achieved using a linear
gradient mode from 150 mM NaOH to 600 mM sodium acetate (in
150 mM NaOH) at the ow rate of 1 mL/min.
2.5. Solubility and swelling power
Starch sample (0.5 g, d.b.) was mixed with DDW (25 mL) in a 50mL centrifuge tube. The aqueous starch suspension was heated for
30 min (with periodically vortexing) at 80 C, and cooled at 15 C
for 20 min. After centrifugation (1500 g, 30 min) of the resulting starch paste, the supernatant were carefully discarded and set
aside. The tube was inverted and allowed to drain for 10 min, followed by precisely measuring the tube weight. Swelling power
(g/g) was calculated as the ratio of weights of the swollen starch

B.-S. Kim et al. / Carbohydrate Polymers 125 (2015) 6168

precipitate to the initial starch sample. For solubility, total sugar

contents within the recovered supernatant were determined using
a sulfuric acidphenol method. Starch solubility was calculated as
the percent ratio of weights of total sugar (converted into glucose)
in the supernatant to initial starch sample.

63

NpAS-treated Sinsunchal rice starch) were negatively correlated to

AM contents of their respective native starches (Pearson correlation
coefcient; r = 0.980, p < 0.01). Consequently, the lowest recovery
was observed for NpAS-treated Goamy rice starch.
3.2. Reaction efciency

2.6. Differential scanning calorimeter (DSC)

Thermal properties of native and NpAS-treated starches (rice
and barley) were determined using a differential scanning
calorimeter (DSC 200 PC Phox, Netzsch, Selb, Germany). Starch
sample (10 mg, d.b.) was weighed directly into a stainless steel
pan, followed by addition of DDW (20 L). The pan was hermetically sealed and equilibrated for 18 h at 25 C. The sample pan was
scanned at a heating rate of 5 C/min over a temperature range of
20140 C. A sealed, empty pan was used as a reference.
2.7. Pasting viscosity
Starch samples (2.24 g, d.b.) were directly weighed into aluminum canisters, followed by addition of DDW to achieve a nal
weight of 28.0 g. Starch suspensions were analyzed using a Rapid
Visco Analyzer (Newport Scientic, NSW, Australia) under continued shear (160 rpm) and the following programmed temperature
prole: beginning with an initial hold at 30 C for 2 min, linear heating to 95 C for 6 min, an intermediate hold at 95 C for 5 min, linear
cooling to 50 C for 5 min, and a nal hold at 50 C for 2 min.
2.8. Statistical Analysis
NpAS treatments were repeated twice for each starch sample. All
measurements were determined at least once for each experimental replicate. All experimental data were analyzed using Analysis
of Variance (ANOVA) and expressed as mean standard deviation.
Signicant differences among mean values were examined by a
Duncans multiple range test at p < 0.05. Statistical computations
and analyses were conducted by SAS software package (version
9.0, SAS Institute, Cary, NC, USA).

Reaction efciency (RE) of NpAS in our reaction system is listed

in Table 1. For comparison of RE in this study to that from the previous study (Kim et al., 2013), the supernatants recovered from the
previous study were analyzed as well. The RE of NpAS reaction
at this scale-up study ranged in 25.031.8% for rice starches and
25.636.1% for barley starches (Table 1), which were 1.21.8 times
enhanced than those (13.727.3% for rice starches and 20.126.9%
for barley starches) in the previous study (Kim et al., 2013). A
dramatic increase in RE was obtained for Goamy, Dongjin, and
Manmi rice starches when compared to 13.7%, 14.8%, and 14.5%,
respectively, from the 35-mL scale reaction. The enhanced RE in
3500-mL scale reaction system may result from mechanical agitation of viscous mixtures of pre-gelatinized starch and sucrose.
Under constant agitation of the reaction mixtures during the reaction, accessibility of NpAS to starch molecules and sucrose would
be enhanced, facilitating its glucosyl transferring activity on nonreducing ends of starch molecules. Meanwhile, the greatest RE was
found with waxy-type Sinsunchal rice and Chal barley starches, corresponding to 31.8% and 36.1%, respectively (Table 1). The RE of rice
starches generally increased as their AM contents decreased. Thus,
waxy-type NpAS-treated rice and barely starches clearly displayed
greater RE than normal counterparts. From this result, it was clearly
shown that AP molecules were more favorable substrate for NpAS
over AM. This relationship might be further attributed to the reaction mixture viscosity of the pre-gelatinized rice starches, which
might increase along with their AM contents. Hagenimana, Pu, and
Ding (2005) showed that the complex viscosities of cold pastes of
rice starches increased with increasing their AM contents from 0.0%
to 29.8%. The native effect of larger AM content on the RE of rice and
barley starches might be partially explained by the restricted accessibility of NpAS to starch molecules and sucrose due to the higher
viscosities of AM-containing pre-gelatinized starch substrates.

3. Results and discussion

3.3. Resistant starch
3.1. Scale-up production of NpAS-treated rice and barley starches
For preparation of NpAS-treated rice and barley starches, the
previous study used a small-scale reaction system with 35 mL of
the substrate mixture with 10 U/mL (total 350 U) of NpAS (Kim
et al., 2013). In this study, the volume of the substrate mixture
was increased up to 3500 mL, but the total NpAS activity was
identical to that in the previous study (Kim et al., 2013). When
the nal yields of NpAS-treated starches were calculated, Goamy,
Dongjin, Manmi, and Sinsunchal rice starches showed 91.5%, 94.4%,
99.7%, and 158.9%, respectively; the production yields of Gwangan and Chal barley starches were 96.4 and 109.3%, respectively.
Within given starch sources, the greatest yields were obtained
from waxy type of rice and barley starches, corresponding to Sinsunchal and Chal varieties, respectively. The NpAS reaction on
waxy starches showed more rapid glucose deposition, from sucrose
as a co-substrate, on non-reducing ends of starch molecules,
which resulted in the exceeding 100% of production yield starting from the initial amount of starch substrate. Meanwhile, the
yields of NpAS-treated rice (Goamy, Dongjin, Manmi) and barley
(Gwangan) starches, possessing considerable amount of amylose
(AM) molecules, ranged from 91.5% to 99.7%. Especially for rice
starches, the yields of NpAS-treated starches were positively correlated to their respective reaction efciencies (Pearson correlation
coefcient; r = 0.995, p < 0.01). Moreover, their yields (except for

NpAS-treated rice and barley starches exhibited RS contents

of 30.250.7% and 43.146.7%, respectively (Table 1), and the
greatest RS contents were obtained from NpAS-treated waxytype Sinsunchal rice (50.7%) and Chal barley (46.7%) starches,
respectively. Generally speaking, RS contents of the NpAS-treated
rice starches increased as AM contents of their respective native
starches decreased. However, a direct correlation between the RS
contents of enzyme-treated products and AM contents of their
native counterparts was not clearly observed RS contents within
NpAS-treated starches generated in this study were 2.03.4 times
greater than those in the previous study (Kim et al., 2013) that the
small volume of the substrate mixture was used for NpAS reaction.
The observed ndings may be due to the improvement of the RE of
the NpAS reaction through more homogenous reaction condition
by constant mechanical agitation. Thus, higher RS contents in the
NpAS-treated starches possessing could be obtained by the scale
up without proportionally increasing the dosage of NpAS from the
previous study.
Considering the relationship between RE and RS contents, the
enhanced RE in 3500-mL (relative to 35-mL) scale reaction system
coincided with the increased RS contents (30.250.7%) of NpAStreated starches (Table 1). RS contents of NpAS-treated starches
in 35-mL scale reaction system of the previous study ranged from
13.6% to 46.1% (Kim et al., 2013). For Goamy, Dongjin, and Manmi

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B.-S. Kim et al. / Carbohydrate Polymers 125 (2015) 6168

Table 1
Reaction efciency and resistant starch content of rice and barley starches treated by NpASA .
NpAS-treated starch

Variety

Reaction efciencyB (%)

Rice

Goamy
Dongjin
Manmi
Sinsunchal
Chal
Gwangan

25.0
25.0
26.3
31.8
36.1
25.6

Barley
A
B

Resistant starch content (%)

0.6d
0.8d
0.3c
0.8b
0.2a
0.2d

36.5
30.2
45.6
50.7
46.7
43.1

0.9e
0.1f
0.4c
0.4a
0.3b
0.9d

Mean values of three replicate measurements; values sharing the same lowercase letters in superscript within columns are not signicantly different at p < 0.05.
Dened as the percent ratio of molar concentrations of fructose in the supernatant recovered after NpAS reaction to initial sucrose concentration (300 mM).

rice starches, the RE in 3500-mL scale reaction system were 1.71.8

times greater than that in 35-mL scale reaction system. These NpAStreated rice starches revealed 2.03.4 times greater RS contents,
and in particular, RS content of NpAS-treated Manmi rice starch
was enhanced to the greatest extent (3.4 times). For Sinsunchal
rice starch and Chal and Gwangan barley starches, however, the RE
in 3500-mL scale reaction system were only 2030% higher than
that in 35-mL scale reaction system (Kim et al., 2013), and the
RS contents of their NpAS-treated ones (3500 mL) were also only
1.01.2 times higher than the small scale reaction system. Thus, RS
contents of NpAS-treated starches were as enhanced to similar or
more degrees as RE increased by scaling up of the reaction process.

Regardless of AM contents within a given starch source, common

patterns were observed for both rice and barley starches that AP A
(DP 612) and B1 (DP 1324) branch-chains decreased, while AP
B2 (DP 2536) and B3 (DP > 37) branch-chains increased (Table 2).
The observed changes in AP branch-chain distributions of NpAStreated starches were also reported by others (Kim et al., 2013; Shin
et al., 2010; Ryu et al., 2010), who demonstrated the lengthening of
AP branch-chains through translocating glucose units from sucrose
onto their non-reducing ends by NpAS treatment. Furthermore, the
pooled value (30.3%) of the decreased ratios in AP A and B1 branchchain proportions (e.g., 27.9% and 2.4%, respectively, for Goamy
rice starch) of NpAS-treated starches relative to native counterparts
approximated the pooled value (30.4%) of the increased ratios in AP
B2 and B3 branch-chain proportions (e.g., 27.0% and 3.4%, respectively, for Goamy rice starch). Overall, the observed phenomena
suggested that reaction of NpAS with starch AP molecules appeared
to predominantly occur at non-reducing ends of AP A and B1 (relative to B2 and B3) branch-chains. This suggestion may be due to the
fact that non-reducing ends of AP A and B1 branch-chains, which
appear to be mainly exposed to the outer surfaces of AP clusters
(Biliaderis, 1982), could have much more chances to react with
NpAS.
To further understand relationship between the lengthening of
AP branch-chains of NpAS-treated starches and their RS contents,
AP branch-chain distributions of NpAS-treated rice and barley
starches were recalled from the previous study (Kim et al., 2013)
which used starch sources identical to those in this study. NpAStreated starches in the previous study exhibited a decreased
proportion of AP A branch-chains and an increased proportion in
AP B1 and B2 branch-chains. In this study, as mentioned earlier,
AP A and B1 branch-chain proportions of NpAS-treated rice and
barley starches decreased, while their AP B2 and B3 branch-chain
proportions increased (Table 2). Also, mean elongation degrees
(determined by a difference in weight-average degree of polymerization between NpAS-treated and native starches) on rice and

3.4. Branch-chain distribution of amylopectin by HPAEC

AP branch-chain length distributions of native and NpAS-treated
starches were analyzed using HPAEC after isoamylolysis, and
the relative ratios of peak areas of their AP branch-chains were
obtained from normalized chromatograms. The proportions of AP
branch-chain categories (i.e., A, B1, B2, and B3 branch-chains)
(Hanashiro, Abe, & Hizukuri, 1996) were calculated from the sum
of the relative peak areas and depicted in Table 2. As reported by
others (Chung, Liu, Lee, & Wei, 2011; Kim et al., 2013; Yoshimoto,
Takenouchi, & Takeda, 2002), number-average and weight-average
degrees of polymerization in APs were almost identical across AM
contents of a given starch source, although very slight variations
were present (Table 2). Also, similar AP branch-chain distributions were observed for starches with different amylose contents
(Table 2), consistent with other reports for rice (Kim et al., 2013)
and barley (Yoshimoto et al., 2002) starches. In contrast, NpAS treatment of pre-gelatinized rice and barley starches in the presence
of sucrose revealed signicant changes in their AP branch-chain
distributions relative to those of native starches (Table 2). Classication of AP branch-chains constructed by Hanashiro et al.
(1996) no longer applied for NpAS-treated rice and barley starches.
Table 2
Amylopectin branch-chain length distribution of native and NpAS-treated starchesA .
Starch source

Variety

Treatment

Rice

Goamy

Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated

DP 612

Dongjin
Manmi
Sinsunchal
Barley

Chal
Gwangan

DPn B

Amylopectin branch-chain distribution

32.5
4.6
30.0
6.4
31.5
2.6
30.9
3.6
27.2
2.7
24.1
2.5

2.1a
1.4b
2.2a
0.3b
0.4a
0.2b
0.8a
0.5b
2.8a
0.5b
4.6a
0.0b

DP 1324
52.9
50.5
54.8
52.2
52.6
44.1
53.0
45.4
50.5
44.8
56.7
52.0

1.4a
4.4a
2.7a
1.5a
3.8a
3.2b
1.5a
0.0b
7.7a
4.6a
6.6a
1.2a

DP 2536
10.4
37.0
10.2
36.7
10.9
44.5
10.2
44.2
16.3
43.3
15.7
38.4

1.6b
0.1a
1.2b
0.4a
0.4b
1.2a
0.2b
0.7a
4.7b
0.0a
0.5b
2.0a

DPw C

DP > 37
4.2
7.1
5.0
4.6
4.9
8.7
5.9
6.6
6.0
9.2
3.4
7.1

0.9a
4.7a
3.7a
2.2a
3.9a
4.5a
0.2a
0.5a
5.5a
5.1a
1.5a
0.7a

14.3
21.1
14.1
20.8
14.1
23.1
14.6
22.7
14.7
22.1
15.8
22.6

0.2b
0.3a
0.2b
0.3a
0.3b
0.1a
0.1b
0.2a
0.3b
0.7a
0.4b
0.1a

17.2
23.7
16.5
23.4
16.3
25.3
17.8
25.2
17.2
24.6
18.5
24.8

0.1b
0.5a
0.2b
0.6a
0.7b
0.3a
0.0b
0.1a
0.1b
0.3a
0.1b
0.1a

A
Mean values of duplicate measurements; values sharing the same lowercase letters in superscript within a given variety of either rice or barley starches are not signicantly
different at p < 0.05.
B
Number-average degree of polymerization.
C
Weight-average degree of polymerization.

B.-S. Kim et al. / Carbohydrate Polymers 125 (2015) 6168

65

Table 3
Solubility and swelling power of native and NpAS-treated starchesA .
Starch source

Variety
Goamy

Rice

Dongjin
Manmi
Sinsunchal
Barley

SolubilityB (%)

Treatment

Chal
Gwangan

45.8
38.5
50.6
46.6
94.5
22.7
119.3
37.2
74.3
23.9
49.5
14.9

Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated

Swelling powerB (g/g)

7.2
4.5
11.2
5.0
12.5
6.4
N/DC
5.4
19.8
4.3
10.7
4.8

2.3
2.5b
1.8a
0.2b
1.8a
2.6b
2.6a
1.2b
6.4a
1.0b
0.7a
0.9b

0.0a
0.1b
0.0a
0.1b
0.0a
0.1b
0.2a
0.0a
0.0b
0.1a
0.2b

A
Mean values of three replicate measurements; values sharing the same lowercase letters in superscript within a given variety of either rice or barley starches are not
signicantly different at p < 0.05.
B
Determined at 80 C.
C
Not detected.

barley starches in this study (DPw 6.39.0) were much greater than
those in the previous study (DPw 2.05.5) (Kim et al., 2013). The
discrepancies in the branch-chain distributions and the degree of
polymerization of AP molecules may result from differences in RE
between reaction systems in this study and the previous study (RE
of NpAS reaction in this study was 1.21.8 time higher than that in
the previous study). As a result of the increase of RE in this study, RS
contents of NpAS-treated starches were enhanced without exception (Table 1). Furthermore, Kim et al. (2013) suggested that AP
A and B1 branch-chain proportions of NpAS-treated starches were
negatively correlated to their RS contents, while the reverse correlation was found for AP B2 and B3 branch-chain proportions and
average degree of polymerization. Overall, the ndings provided
strong evidence that the greater lengthening of starch AP branchchains by NpAS treatment resulted in the enhanced RS contents of
NpAS-treated starches (Table 1).

2011; Vandeputte, Derycke, Geeroms, & Delcour, 2003). In contrast,

solubilities and swelling powers of NpAS-treated rice and barley
starches were lower than those of their respective native counterparts (Table 3), and did not appear to be inuenced by the AM
contents of their parent starches, different from the reports in literature (Chung et al., 2011; Tester & Morrison, 1993; Vandeputte
et al., 2003). The noted phenomena may be explained by the AP
branch-chain distribution of NpAS-treated starches and the suggestion of Srichuwong, Sunarti, Mishima, Isono, and Hisamatsu
(2005) that lack of short AP branch-chains (DP 612) (relative
to the intermediate AP chains) would increase the stability of
the crystalline packing, retard penetration of water into granule
(hydration), and in turn prevent granule swelling. In this study,
also, the lengthening of starch AP branch-chains by NpAS treatment resulted in the deciencies in AP A (DP 612) and B1 (1324)
branch-chains and the enrichments in AP B2 and B3 branch chains
of NpAS-treated starches (Table 2), which might enhance the probability of forming stable recrystallized AP clusters by aggregation
among the lengthened AP branch-chains (Srichuwong et al., 2005).
Accordingly, the possibly stabilized crystalline structures of NpAStreated starches would restrict their hydration and starch molecule
mobility, reducing their solubilities and swelling powers. Another
possible explanation would be that NpAS-treated starches have
already lost their granularities by pre-gelatinization of native starch
granules prior to NpAS reaction. Swelling of starch granules is the
process to incorporate water within starch granules while the rigidity of swollen starch granules could be maintained (BeMiller, 1997,
chap. 6). Therefore, NpAS-treated starches which have already
lost their granular form by cooking prior to NpAS reaction would

3.5. Solubility and swelling power

Solubility and swelling power of NpAS-treated rice and barley
starches were determined at 80 C (Table 3), because the supernatants and swollen starches were not able to be separated by
centrifugation from starch-water suspensions heated at 90 C, in
the cases of Sinsunchal and Manmi rice starches, and both barley starches. For comparison, the solubility and swelling power
of native rice and barley starches as controls were also investigated at 80 C (Table 3). Solubilities and swelling powers of native
rice and barley were enhanced with decreasing AM contents of
starches (Table 3), consistent with other reports (Chung et al.,

Table 4
Thermal transition properties of native and NpAS-treated starchesA .
Starch

Variety

Treatment

To ( C)B

Rice

Goamy

Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated
Native
NpAS-treated

58.6
87.3
61.5
84.6
63.8
86.2
62.8
75.6
61.1
76.3
62.6
76.2

Dongjin
Manmi
Sinsunchal
Barley

Chal
Gwangan

Tp ( C)B
0.1b
0.9a
0.1b
0.4a
0.4b
0.1a
0.7b
1.9a
0.1b
0.3a
0.0b
0.6a

64.7
99.3
68.0
95.2
69.8
97.4
70.3
95.0
66.1
95.9
67.0
95.9

Tc ( C)B
0.0b
0.6a
0.5b
0.1a
0.6b
0.3a
0.8b
0.8a
0.0b
2.3a
0.1b
0.8a

71.7
113.2
73.5
104.0
75.8
111.3
78.6
109.6
71.9
102.2
73.7
102.8

HB (J/g)
0.2b
0.1a
0.5b
0.3a
0.9b
1.0a
0.5b
3.4a
0.1b
0.1a
0.1b
0.4a

8.1
10.7
6.4
6.6
6.5
9.6
7.0
15.7
9.0
7.7
6.7
7.8

0.7b
0.3a
0.7a
0.6a
0.2b
0.7a
0.6b
3.2a
0.4b
0.7a
0.3b
0.3a

A
Mean values of duplicate measurements; values sharing the same lowercase letters in superscript within a given variety of either rice or barley starches are not signicantly
different at p < 0.05.
B
To , Tp , and Tc refer to onset, peak, and completion temperatures, respectively, for the gelatinization of native starches and the melting of NpAS-treated starches. H
indicates gelatinization and melting enthalpies for native and NpAS-treated starches, respectively.

66

B.-S. Kim et al. / Carbohydrate Polymers 125 (2015) 6168

Fig. 1. Pasting viscosity proles of native (solid lines) and NpAS-treated (dotted lines) starches (A, Goamy rice; B, Dongjin rice; C, Manmi rice; D, Sinsunchal rice; E, Chal
baley; F, Gwangan barley).

B.-S. Kim et al. / Carbohydrate Polymers 125 (2015) 6168

exhibited lower capacities to hold water within NpAS-treated

starches. Consequently, their lower solubilities and swelling powers may be explained by both suggested mechanisms.

67

NpAS-treated starches were commonly observed for cross-linked

starches (Kim, Hwang, Kim, & Baik, 2012; Koo, Lee, & Lee, 2010).
4. Conclusions

3.6. Thermal properties

For rice and barley starches, the gelatinization properties of
native starch and the melting properties of NpAS-treated starches
were analyzed using DSC (Table 4). Although gelatinization and
melting properties of native and NpAS-treated starches, respectively, were not directly related to starch AM contents (Table 4),
it was still valid to investigate effects of NpAS treatment on thermal properties of rice and barley starches. Melting temperatures of
NpAS-treated rice and barley starches were shifted upward relative
to gelatinization temperatures of their respective native starches
(Table 4). Melting temperature ranges (Tc -To ) of NpAS-treated
starches (15.826.6 C) were broader than gelatinization temperature ranges of their respective native starches (10.815.8 C).
Melting enthalpies were generally higher for NpAS-treated rice
and barley starches (except for Chal barley starch) than gelatinization enthalpies of their native counterparts (Table 4). Tester and
Morrison (1990) noted that gelatinization temperatures of native
starches are associated with their degree of crystallite perfection
and stability, while gelatinization enthalpy indicates the overall
degree of crystallinity. Similarly, the increased melting temperatures of NpAS-treated starches suggested that NpAS treatment may
improve the perfection of their crystallites. Further, the broader
melting temperature ranges and higher melting enthalpies may
indicate the presence of crystallites possessing a wide range of crystallite perfection within NpAS-treated starches. Furthermore, these
suggestions were consistent with the idea that the stable crystalline
structures formed from re-association among the lengthened AP
branch-chains by NpAS treatment may restrict both solubility and
swelling power of NpAS-treated starches as mentioned earlier.
3.7. Pasting viscosity
Pasting viscosity proles of native and NpAS-treated starches
were investigated using the RVA, and shown in Fig. 1. However,
their pasting viscosity parameters were not depicted, due to the
distinct pasting viscosity proles of NpAS-treated starches. Native
rice and barley starches exhibited typical pasting viscosity proles
as reported by others (Chung et al., 2011; Yoshimoto et al., 2002).
However, NpAS-treated starches revealed lower viscosities across a
temperature prole than their native counterparts. Pasting temperatures (84.191.3 C) of NpAS-treated starches were much higher
than those (65.478.3 C) of their respective native starches, coincident with shifts in melting onset temperature of NpAS-treated
starches to higher degrees (Table 4). These results were consistent
with the report of Ryu et al. (2010), who showed lower pasting
viscosity and higher pasting temperature of NpAS-treated (relative to native) normal corn starch. The noted reduction in pasting
viscosities and increase in pasting temperatures of NpAS-treated
starches were due to their reduced solubilities and swelling powers (Table 3) resulting from their recrystallized (further stabilized
through the lengthening of AP molecules by NpAS treatment) and
non-granular structures. In general, pasting viscosities by RVA were
obtained from a serial phase transition (hydration, swelling, fragmentation of swollen granules, and re-association of leached starch
molecules) of starch granules under a programmed temperature
prole and constant stirring (BeMiller, 1997, chap. 6). However,
NpAS-treated (relative to native) starches possessing much lower
solubilities and swelling powers may not undergo the serial phase
transition occurring in the RVA analysis, which failed to develop the
enhanced pasting viscosities. The similar phenomena observed for

This study investigated characteristics of NpAS-treated rice and

barley starches reacted with relatively lower NpAS activity per unit
volume than used previously. An increase of RE in NpAS reaction by
scale-up enhanced RS contents within NpAS-treated starches. The
lengthened AP branch-chains (less AP A and B1 branch-chains and
more AP B2 and B3 branch chains) by NpAS treatment might derive
stable crystalline structure within NpAS-treated starches, resulting
in their reduced solubilities and swelling powers. For NpAS-treated
starches, on the other hand, the reduced solubilities and swelling
powers, increased melting properties, reduced pasting viscosities,
and enhanced RS resembled general characteristics observed for
phosphorylated cross-linked starches. Accordingly, NpAS-treated
starches prepared in this study may possess the potential to replace
commercial RS products prepared by phosphorylated cross-linking
reaction.
Acknowledgement
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MEST) (No.
2012R1A2A2A01047497).
References
BeMiller, J. N. (1997). Carbohydrate chemistry for food scientists ((2nd ed.)). St. Paul:
AACC International Press.
Biliaderis, C. G. (1982). Physical characteristics, enzymatic digestibility, and structure of chemically modied smooth pea and waxy maize starches. Journal of
Agricultural and Food Chemistry, 30, 925930.
Chung, H.-J., Liu, Q., Lee, L., & Wei, D. (2011). Relationship between the structure,
physicochemical properties and in vitro digestibility of rice starches with different amylose contents. Food Hydrocolloids, 25, 968975.
Chung, H.-C., Woo, K.-S., & Lim, S.-T. (2004). Glass transition and enthalpy relaxation
of cross-linked corn starches. Carbohydrate Polymers, 55, 915.
Hagenimana, A., Pu, P., & Ding, X. (2005). Study on thermal and rheological properties of native rice starches and their corresponding mixtures. Food Research
International, 38, 257266.
Hanashiro, I., Abe, J., & Hizukuri, S. (1996). A periodic distribution of the chain length
of amylopectin as revealed by high-performance anion-exchange chromatography. Carbohydrate Research, 283, 151159.
Higgins, J. A. (2004). Resistant starch: Metabolic effects and potential health benets.
Journal of AOAC International, 87, 761768.
Kim, B.-S., Kim, H.-S., Hong, J.-S., Huber, K. C., Shim, J.-H., & Yoo, S.-H. (2013). Effects
of amylosucrase treatment on molecular structure and digestion resistance of
pre-gelatinised rice and barley starches. Food Chemistry, 138, 966975.
Kim, H.-S., & BeMiller, J. N. (2012). Effects of hydrocolloids on the pasting and paste
properties of commercial pea starch. Carbohydrate Polymers, 88, 11641171.
Kim, H.-S., Hwang, D.-K., Kim, B.-Y., & Baik, M.-Y. (2012). Cross-linking of corn starch
with phosphorus oxychloride under ultra high pressure. Food Chemistry, 130,
977980.
Kim, N.-H., Kim, J.-H., Lee, S., Lee, H., Yoon, J.-W., Wang, R., & Yoo, S.-H. (2010).
Combined effect of autoclaving-cooling and cross-linking treatments of normal
corn starch on the resistant starch formation and physicochemical properties.
Starch/Starke, 62, 358363.
Koo, S. H., Lee, K. Y., & Lee, H. G. (2010). Effect of cross-linking on the physicochemical
and physiological properties of corn starch. Food Hydrocolloids, 24, 619625.
Lee, K. W., Yoo, S.-H., & Lee, H. G. (2012). The effect of chemically-modied resistant
starch RS type-4, on body weight and blood lipid proles of high fat diet-induced
obese mice. Starch/Starke, 64, 7885.
McCleary, B. V., & Monaghan, D. A. (2002). Measurement of resistant starch. Journal
of AOAC International, 85, 665675.
Ryu, J.-H., Lee, B.-H., Seo, D.-H., Baik, M.-Y., Park, C.-S., Wang, R., & Yoo, S.-H. (2010).
Production and characterization of digestion-resistant starch by the reaction of
Neisseria polysaccharea amylosucrase. Starch/Starke, 62, 221228.
Sharma, A., Singh, B., & Ritika, Y. (2008). Resistant starch: Physiological roles and
food applications. Food Reviews International, 24, 193234.
Shin, H. J., Choi, S. J., Park, C. S., & Moon, T. W. (2010). Preparation of starches with low
glycemic response using amylosucrase and their physicochemical properties.
Carbohydrate Polymers, 82, 489497.
Srichuwong, S., Sunarti, T. C., Mishima, T., Isono, N., & Hisamatsu, M. (2005). Starches
from different botanical sources II: Contribution of starch structure to swelling
and pasting properties. Carbohydrate Polymers, 62, 2534.

68

B.-S. Kim et al. / Carbohydrate Polymers 125 (2015) 6168

Tester, R. F., & Morrison, W. R. (1990). Swelling and gelatinization of cereal starches.
I. Effects of amylopectin, amylose and lipids. Cereal Chemistry, 67, 551557.
Tester, R. F., & Morrison, W. R. (1993). Swelling and gelatinization of cereal starches
III. Some properties of waxy and normal nonwaxy barley starches. Cereal Chemistry, 69, 654658.
Vandeputte, G. E., Derycke, J., Geeroms, J., & Delcour, J. A. (2003). Rice starches. II.
Structural aspects provide insight into swelling and pasting properties. Journal
of Cereal Science, 38, 5359.
Villwock, V. K., & BeMiller, J. N. (2005). Effects of salts on the reaction of normal corn
starch with propylene oxide. Starch/Starke, 57, 281290.

Woo, K., & Seib, P. A. (2002). Cross-linked resistant starch: Preparation and properties. Cereal Chemistry, 79, 819825.
Wang, R., Bae, J.-S., Kim, J.-H., Kim, B.-S., Yoon, S.-H., Park, C.-S., & Yoo, S.H. (2012). Development of an efcient bioprocess for turanose production
by sucrose isomerisation reaction of amylosucrase. Food Chemistry, 132,
773779.
Yoshimoto, Y., Takenouchi, T., & Takeda, Y. (2002). Molecular structure and some
physicochemical properties of waxy and low-amylose barley starches. Carbohydrate Polymers, 47, 159167.